The principal aim of this proposal is to study the selectivity of connections formed in vitro by individual, identified neurons. Cells isolated from the CNS of the medicinal leech offer distinct advantages for this study. A wide variety of sensory neurons, motor neurons and interneurons have been identified in leech ganglia. The cells are easily isolated using a simple mechanical technique. They survive well, sprout and can form chemical and electrical connections in culture. The strategy of this project will be to isolate identified neurons, place them together in pairs in vitro and record from the pairs to determine whether a connection has formed between the cells. This procedure will be repeated to test pairwise combinations of the broad spectrum of neurons available in the leech and thereby construct a detailed picture of the preferences of the cells for synaptic partners. Initial experiments show that the formation of connections in culture can be selective: Retzius cells electrically couple with other Retzius cells when paired in vitro, but not with pressure sensory P cells. The anatomy and physiology of the cells and their connections formed in vitro will be studied and compared with their in vivo counterparts. Particular attention will be paid to the anatomical and physiological events surrounding the initial encounter between cells. In vivo studies of neurospecificity have often been limited by the complexity of nervous tissue; in vitro studies by the difficulty of unambiguously identifying neurons dissociated from heterogeneous tissue. The present approach overcomes these obstacles by combining the relative simplicity and control of tissue culture with the advantages of identified neurons. The synaptic preferences revealed in these experiments could provide an assay to test the effects of biochemical and immunological manipulations on the ability of neurons to recognize appropriate targets and thus provide insights into the mechanisms of neurospecificity.